This year has seen the continued production of the recombinant galectin-1 homodimer, a C2S mutant containing an N-terminal poly-His tag. This material was used to explore solution conditions and obtain a variety of 1H-NMR spectra, either with the protein alone or in the presence of known oligosaccharide ligands. Binding studies with oligosaccharides with up to three repeating lactosamine units using NMR spectroscopy have shown a picture consistent with published X-ray structures and other competitive binding studies. A terminal galactosyl, if present, is the favored region of interaction, and N-acetyllactosamine regions are favored over lactose regions. We currently have [13C]-labeled glucosamine and [13C]-labeled glucose which are being incorporated into these oligosaccharides as N-acetylglucosamine and galactose, respectively. The labeled ligands will greatly facilitate the collection of structurally useful NMR data, such as inter- and intramolecular NOE measurements and heteronuclear coupling constants. An important step towards the complete NMR description of this protein-carbohydrate system was the production of approximately 10 mg of [15N]-galectin-1. {1H-15N} correlated spectra have been obtained, and multidimensional experiments are in progress. These spectra will be used to study amide proton and nitrogen shifts resulting from ligand binding and will aid in the assignment procedure. Due to the high molecular weight (~30 kD) of the dimer, full assignment will probably only be possible with the additional [13C]-label and the use of triple-resonance multidimensional NMR techniques. A different approach is also being explored that involves producing galectin-1 mutants that allow for high concentrations of stable, active monomers. Primers were designed with deletions at the C- and N-termini, which are both involved in the dimer interaction. Using standard methods, E. coli was transformed with pQE-60 vectors containing the cDNA insert and induced. The isolated monomer is currently being studied for binding activity. If successful, the monomer would fall in the molecular weight range where resonance assignment is much easier, perhaps avoiding the need for expensive [13C]-labeling. In addition, further NMR data on ligand binding and dynamics and computational and thermodynamic studies of the complex would be easier to interpret. It is well established that the best interpretation of NMR data for conformational studies is dependent on suitable computational models. Dr. Woods has been exploring the binding of a poly-N-acetyllactosamine heptamer to galectin-1 using molecular dynamics. The calculated bound structures can provide theoretical NOE and heteronuclear J-coupling values with which to compare the same experimentally obtained NMR parameters.